ABSTRACT To define the (co-)expression pattern of target receptor-tyrosine-kinases (RTK) in human gastric adenocarcinoma.
The (co-)expression pattern of VEGFR1-3, PDGFR alpha/beta and EGFR1 was analyzed by RT-PCR in 51 human gastric adenocarcinomas. In addition, IHC staining was applied for confirmation of expression and analysis of RTK localisation.
The majority of samples revealed a VEGFR1 (98%), VEGFR2 (80%), VEGFR3 (67%), PDGFR alpha (82%) and PDGFR beta (82%) expression, whereas only 62% exhibited an EGFR1 expression. 78% of cancers expressed at least four out of six RTKs. While VEGFR1-3 and PDGFR alpha revealed a predominantly cytoplasmatic staining in tumor cells, accompanied by an additional nuclear staining for VEGFR3, EGFR1 was almost exclusively detected on the membrane of tumor cells. PDGFR beta was restricted to stromal pericytes, which also depicted a PDGFR alpha expression.
Our results reveal a high rate of receptor-tyrosine-kinases coexpression in gastric adenocarcinoma and might therefore encourage an application of multiple-target RTK-inhibitors within a combination therapy.

or Ras[12,13]. Expression of functional receptors and their respective ligands by tumor cells also raises the possibility of autocrine loops and is critical for auto-stimulation and progression[14,15]. Hitherto, the growth factors VEGF and PDGF and their receptors have been considered relevant in the proc-ess of angiogenesis and dissemination in gastric adenocar-cinoma, whereas EGF/EGFR was correlated with tumor growth and local invasion[16-20]. As part of the tyrosine kinase family, PDGF receptors are involved in multiple tu-mor-associated processes, like enhancing tumor angiogen-sis by recruitment and regulation of tumor fibroblasts and pericytes[21]. Data correlating PDGFRα or PDGFRb expression with clinical outcome in human gastric adeno-carcinoma are to the best of our knowledge not available. As new multi-target tyrosine kinase inhibitor are emerg-ing and enriching the therapy in various malignancies, our aim was to define the expression pattern of target RTKs in human gastric adenocarcinoma and thus elucidate a ra-tionale for a possible new therapeutic strategy[22,23].MATERIALS AND METHODSTissue source and storageTumour samples were obtained from 51 patients undergoing elective surgery for gastric adenocarcinoma at the University of Mainz between 2005 and 2006. Specimens were conventionally stored in formalin for consecutive histopathological analysis. In addition to conventional processing of tissues in formalin for standard analysis, small samples of each specimen were stored in cryovials, shock frozen in liquid nitrogen immediately after extirpation and stored at -80℃ until further processing. Those tumour tissue originated from the centre of the tumour. As control tissues, samples of healthy gastric mucosa were collected from the margin of gastrectomy specimens, performed due to non-malignant disease. Informed consent was obtained before the respective tissue was collected. ImmunohistochemistryParaffin-embedded tissue samples of normal gastric mucosa and gastric adenocarcinoma were generously provided by S Biesterfeld the Institute of Pathology, University of Mainz and were screened for VEGFR1-3, PDGFRα /b and EGFR1 protein expression by immunohistochemistry (Table 1). The tissues were deparaffinized, rehydrated and subsequently incubated with the respective primary antibody (Table 1). The secondary antibody (anti-rabbit-mouse-goat-antibody) was incubated for 15 min at room temperature, followed by an incubation with strepavidin-POD (DAKO, Germany) for 15 min. Antibody binding was visualized using AEC-solution (Dako, Germany). Finally, the tissues were counterstained by haemalaun solution (DAKO, Germany). RNA isolation and RT-PCRRNA isolation was performed using the RNeasy Kit according to the manufacturer's recommendations (Qiagen, Hilden, Germany). Transcription of b-Actin, VEGFR1, VEGFR2, VEGFR3, PDGFRα, PDGFRb and EGFR1 was analyzed by a two-step RT-PCR: reverse transcription was performed with 2 μg of RNA (20 μL total volume; Omniscript RT Kit, Qiagen) according to the recommendations of the manufacturer. In total, 0.5 μL of the cDNA (50 ng) were used as template for the specific PCR reactions. Primers applied were b-Actin-forward: 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3' and reverse 5'-CTA GAA GCA TTT GCG GTG GAC GAC GGA GGG-3' (661 base pairs (bp) fragment), VEGFR1-forward: 5'-TGG GAC AGT AGA AAG GGC TT-3' and reverse: 5'-GGT CCA CTC CTT ACA CGA CAA-3' (394 bp), VEGFR2-forward: 5'-CAT CAC ATC CAC TGG TAT TGG-3' and reverse: 5´-GCC AAG CTT GTA CCA TGT GAG-3' (400 bp), VEGFR3-forward: 5'-CCC ACG CAG ACA TCA AGA CG-3' and reverse: 5'-TGC AGA ACT CCA CGA TCA CC-3' (380 bp), PDGFRα-forward: 5'-CTC CTG AGA GCA TCT TTG AC-3' and reverse 5'-AAG TGG AAG GAA CCC CTC GA-3', PDGFRb-forward: 5'-TCC TCA ATG TCT CCA GCA CCT TC-3' and reverse 5'-ACC ACA GTC TGC ACT GCG TTC-3' (547 bp) and EGFR1-forward: 5'-TCT CAG CAA CAT GTC GAT GGA-3' and reverse: 5’CGC ACT TCT TAC ACT TGC GG-3' (474 bp). For amplification a DNA Engine PTC200 (MJ Research, Watertown, MA) thermocycler was used. Cycling conditions of the respective PCRs were as follows: intitial denaturation (4 min at 94℃) followed by the respective number of cycles (b-Actin: 28, VEGFR1: 36, VEGFR2: 38, VEGFR3: 38, PDGFRα: 40, PDGFRb : 36 and EGFR1: 38) of denaturation (1 minute at 94℃), annealing (45 s; b-Actin: 52℃, VEGFR1: 60℃, VEGFR2: 62℃, VEGFR3: 62℃, PDGFRα: 57℃, PDGFRb: 64℃ and EGFR1: 60℃) and elongation (1 minute at 72℃). After the last cycle a final extension (7 min at 72℃) was added and thereafter the samples were kept at 4℃. Seven μL of the product were run on a 1.8% agarose gel, stained by ethidium bromide, and analyzed under UV light. The evaluation of expression was performed semiquantitatively according to following grades: negative: 0, weak: 1, medium: 2, strong: 3.RESULTSImmunohistochemical staining of receptor tyrosine kinases in gastric mucosa and adenocarcinoma Negative controls of gastric mucosa and adenocarcinoma Table 1 Antibody charcteristicsTarget AntibodyDilution IncubationVEGFR1Flt-1 (C-17)1:1004 hSanta Cruz Biotechnology, CA, USASanta Cruz Biotechnology, CA, USASanta Cruz Biotechnology, CA, USACell Signalling Technology, MA, USABioGenex, CA, USAVEGFR3 Flt-4 (C20)1:200 2 hPDGFRαPDGFRα (C-20)1:200 2 hPDGFRbPDGFRb (28 E1) 1:2002 hEGFR1AM207-5ME 1:11 h3606 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol July 14, 2007 Volume 13 Number 26www.wjgnet.com

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remained negative for all samples. RTK expression in healthy gastric mucosa varied from strong (EGFR) to intermediate (VEGFR2) and barely detectable/absent (VEGFR1, VEGFR3, PDGFRα ; PDGFRb , Figure 1A). Analyses by PCR revealed an additional VEGFR1 amplicon which could be ascribed to endothelial cells by IHC staining (Figure 1A). Cancer cells stained for VEGFR1, VEGFR2, VEGFR3, PDGFRα and EGFR1, whereas stromal cells stained for PDGFRα and PDGFRb. VEGFR1, VEGFR2, VEGFR3 and PDGFRα /b revealed a predominantly cytoplasmatic as well as a weak membranous localisation, whereas EGFR1 was almost exclusively fond on the membrane (Figure 1B). An additional nuclear staining was only seen for VEGFR3. Receptor tyrosine kinase expression patterns VEGFR1, VEGFR2, VEGFR3, PDGFRα, PDGFRb and EGFR1 expression in gastric adenocarcinoma samples re-vealed varying transcription intensities. VEGFR1 expres-sion was observed in 98% of all samples and varied from strong (50%) to intermediate (34%) and weak (16%; Figure 2A). VEGFR2 expression was found in 80% of all gastric carcinoma specimens and ranged from weak (39%), to in-termediate (15%) and strong (46%). The overall expression rate of VEGFR3 was 67% with a weak expression in 21%, an intermediate expression in 35% and a strong expression in 44%. PDGFRα expression was observed in 82% of all samples. A strong PDGFRα-expression was found in 50%, whereas 29% revealed an intermediate and 21% a weak ex-pression. PDGFRb expression was seen in 82% and varied from weak (29%) to intermediate (29%) and strong (42%). The expression rate of EGFR1 was 62% and varied from weak (28%), to intermediate (36%) and strong (36%). Receptor tyrosine kinase co-expression and correlation with clinicopathological parameters 36% of samples revealed a coexpression of 6 receptors, 28% of 5 receptors, 14% of 5 receptors and only 34% showed co-expression of 3 receptors or less (Figure 2B). Co-expression of VEGFR1, VEGFR2 and VEGFR3 was found in 63% of samples. DISCUSSIONThis is the first study analyzing the (co-)expression profile VEGFR1 VEGFR2 VEGFR3 PDGFRα PDGFRb EGFR1Mucosa sampleATumor tissuePatient No 10 VEGFR1 PDGFRα VEGFR2 PDGFRb VEGFR3 EGFR1Patient No 1 2 3 4 5 6 7 8 9 10VEGFR1VEGFR2VEGFR3PDGFRαPDGFRbEGFR1b-actinBFigure 1 A: The IHC staining of healthy gastric mucosa for VEGFR1-3, PDGFR-α/b and EGFR1. While healthy gastric mucosa revealed a significant EGFR1 and an intermediate VEGFR2 expression in gastric epithelial cells, all other RTKs exhibited only faint to absent expression levels; B: The exemplary transcription profile of 10 gastric cancers and an immunohistochemical analyses of an adenocarcinoma specimen originating from one patient. While VEGFR1, VEGFR2, VEGFR3 and PDGFRα revealed a predominantly cytoplasmatic staining accompanied by an additional nuclear staining for VEGFR3, EGFR1 was almost exclusively restricted to the membrane of tumor cells. Stromal cells revealed expression of PDGFRα and PDGFRb.Drescher D et al. RTK expression in gastric cancer 3607www.wjgnet.com

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of a series of receptor tyrosine kinases in human gastric adenocarcinoma. We initiated this study while a series of novel multi-target RTK-inhibitors are emerging and enriching classical chemotherapy strategies in order to estimate the benefit of such a therapy in gastric cancer. Our analysis was based on the assumption that tumors co-expressing multiple RTKs are functionally more dependent on ligand binding and more prone to deprivation of those stimuli. RTKs most frequently targeted by available small molecules were chosen for this analysis.VEGFR1-3, PDGFRα/b and EGFR1 undergo phos-phorylation following ligand binding resulting in tyro-sine kinase activity and concomitant activation of RAS-Raf-MEK1/2-ERK1/2 pathways[12,13]. Depending on the location of the RTK on tumor cells, endothelial cells or pericytes, the consequences are tumor cell proliferation, dissemination or angiogenesis. VEGFR1 and VEGFR2 are expressed on endothelial cells, whereas VEGFR3 is largely restricted to lymphatic endothelial cells. While VEGFR1 expression in gastric adenocarcinoma has been associated with tumor prolifera-tion and dissemination[24], VEGFR3 expression has been correlated with lymphatic dissemination in gastric adeno-carcinoma[17]. In a recent study, VEGFR2 expression did neither correlate with prognosis or dissemination in gastric adenocarcinoma[25]. Several studies have proven the impact of EGFR1 expression on poor survival not only in gas-tric adenocarcinoma [20,26]. PDGFRα expression has been described by Tsuda and colleagues in 8 out of 15 gastric adenocarcinomas analyzed, supporting our results[27]. No further groups have reported on PDGFRα expression in gastric adenocarcinoma, since. Data correlating the PDGFRα expression with clinical parameters in gastric adenocarcinoma are not available. However, PDGFRα expression and activating mutations have been reported in gastrointestinal stromal tumors (GIST)[28]. Most interest-ingly, PDGFRα mutated GIST displayed an epitheloid or mixed phenotype and were exclusively located in the stom-ach, whereas PDGFRα wildtype tumors also occurred in the small bowel [29]. PDGFRb expression in gastric adenocarcinoma was only described once in 1992 by Chung and colleagues analyzing three tumor samples[30]. As to the best of our knowledge, no data are available correlating PDGFRb ex-pression with the clinical outcome of patients. However, in other tumor entities such as Ewing sarcoma and breast cancer, PDGFRα and PDGFRb expression strongly cor-related with an invasive behaviour[31,32]. So far, analyses of coexpression patterns in human malignancies have not been performed previously. Among our patients the majority of gastric adenocarcinoma sam-ples revealed an expression of VEGFR1-3 and PDGFRα/ PDGFRb, whereas two thirds exhibited an EGFR1 expres-sion.As expected, PDGFRα and PDGFRb staining was de-tected in the tumor stroma and can most likely be allocated to pericytes. While PDGFRb was restricted to stromal cells only, PDGFRα expression could also be detected in cancer cells. In contrast, VEGFR1-3 and EGFR1 expression was restricted to tumor cells. Hence, tumor cells might engage these RTKs in order to emerge to a proliferative and mi-gratory phenotype.PCR analyses might also amplify RTKs transcribed by vascular and lymphatic endothelial cells or pericytes in the tumor bed. However, as RTK-inhibitors target RTKs not only on cancer cells, but also on endothelial cells and peri-cytes, differentiation of the origin of RTK-transcription might be unneeded as RTK-inhibitors will impact on the tumor bed as total. Endocytosis and translocation of RTKs to the perinu-clear rab4-, rab5- and rab11-compartments of receptor re-cycling has previously been reported for diverse membrane receptors helping to explain the cytoplasmatic location as a consequence of receptor-activation[33,34]. In addition, VEGFR2 is held in an endosomal storage pool and can be delivered to the plasma membrane upon induction [35].Most strikingly, 64% of all cancers analyzed expressed at least five out of six receptor-tyrosine-kinases. Thus, the majority of cases revealed a high frequency of RTK co-expression. In contrast, only 2% of the specimens exhibited no RTK expression at all. Co-expression of VEGF receptors 1-3 was found in 63% of all samples, indicating a relevant role of the vascular and lymphatic endothelial growth factor system in gastric adenocarcinoma. In summary, we report the first coexpression profile of target receptor-tyrosine-kinases in a solid human malignancy.These results might encourage the application of mul-tiple-targeted RTK-inhibitors in gastric adenocarcinoma as part of a multidrug therapy. In order to evaluate the impact of multi-targeted RTK-inhibitors we currently ran a phase Ⅱ study analyzing the benefit of Sunitinib [36], tar-geting the above analyzed RTKs in CPT-11 or Cisplatin re-A VEGFR1 2%18% VEGFR2 20% VEGFR333%98% 80%67% PDGFRα PDGFRb 18% EGFR138%82%82%62%B2% no receptor1 receptor 2%36% 6 receptors28% 5 receptors4 receptors 14%3 receptors 6%2 receptors 12%Figure 2 A: The expression profile of RTKs VEGFR1-3, PDGFRα/b and EGFR1 in human gastric adenocarcinoma; B: The co-expression rates of those RTKs.3608 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol July 14, 2007 Volume 13 Number 26www.wjgnet.com

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[Show abstract][Hide abstract]ABSTRACT:
Despite advances in the treatment of gastric cancer, it remains the world's second highest cause of cancer death. As gastric cancer is often diagnosed at an advanced stage, systemic chemotherapy is the mainstay of treatment for these patients. However, no standard palliative chemotherapy regimen has been accepted for patients with metastatic gastric cancer. Palliative chemotherapy including fluoropyrimidine, platin compounds, docetaxel and epirubicin prolongs survival, and improves a high quality of life to a greater extent than best supportive care. The number of clinical investigations associated with targeted agents has recently increased. Agents targeting the epidermal growth factor receptor 1 and human epidermal growth factor receptor 2 (HER2) have been widely tested. Trastuzumab was the first target drug developed, and pivotal phase III trials showed improved survival when trastuzumab was integrated into cisplatin/fluoropyrimidine-based chemotherapy in patients with metastatic gastric cancer. Trastuzumab in combination with chemotherapy was thus approved to be a new standard of care for patients with HER2-positive advanced esophagogastric adenocarcinoma. Thus, the evaluation of HER2 status in all patients with metastatic gastroesophageal adenocarcinoma should be considered. Other agents targeting vascular endothelial growth factor, mammalian target of rapamycin, and other biological pathways have also been investigated in clinical trials, but showed little impact on the survival of patients. In this review, systemic chemotherapy and targeted therapies for metastatic gastric cancer in the first- and second-line setting are summarized in the light of recent advances.

[Show abstract][Hide abstract]ABSTRACT:
Some tyrosine kinase receptors (RTKs) play critical roles in gastric cancer progression. Not only trastuzumab, but also several other agents targeting RTKs are being investigated for gastric cancer therapy. However, the simultaneous expression of multiple RTKs, which may interfere with the effectiveness of therapeutic agents, has not been evaluated in a large cohort with gastric adenocarcinoma (GAC).
We performed a tissue microarray analysis in 950 patients with GAC who underwent a gastrectomy without preoperative chemotherapy. The protein expressions of HER2, EGFR, MET and FGFR2 were evaluated using immunohistochemistry, and the gene amplifications of HER2, EGFR and MET were examined using dual-color in situ hybridization.
The frequency of overexpression was 11.8 % for HER2, 23.5 % for EGFR, 24.9 % for MET and 31.1 % for FGFR2. Whereas strong staining for each of the RTKs was heterogeneous, tumors with homogeneously strong staining areas often exhibited gene amplification. Strong EGFR expression was significantly associated with a poor outcome, but no prognostic correlations were observed in other RTKs. The overexpression of single and multiple RTKs was observed in 40.4 and 22.7 % of the cases, respectively. HER2, EGFR, MET and FGFR2 predominance was observed in 10.1, 13.9, 16.1 and 22.9 % of the GACs, respectively.
Approximately two-thirds of patients with GAC exhibited the expression of at least one RTK and would be candidates for targeted therapies. Moreover, one-third of at least one RTK overexspressing cases showed multiple RTKs expression. Our results may be useful for selecting the most suitable patients for each targeted therapy.

[Show abstract][Hide abstract]ABSTRACT:
Combination of fluoropyrimidines and a platinum derivative are currently standards for systemic chemotherapy in advanced adenocarcinoma of the stomach and gastroesophageal junction (GEJ). Nevertheless, individual likelihood for response to these therapeutic regimes remains uncertain. Even more, no predictive markers are available to determine which patients may benefit more from oxaliplatin versus cisplatin or vice versa. The new invasion and stem cell markers VEGFR-3 and CXCR4 have been linked prognostically with more aggressive esophagogastric cancer types. Thus, we aimed to assess correlations of VEGFR-3 and CXCR4 expression levels with clinical outcome in a randomized phase III study of patients with oxaliplatin/leucovorin/5-FU (FLO) versus cisplatin/leucovorin/5-FU (FLP).